Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development.
Commonly used methods for the determination of protein binding, e.g. ultrafiltration or equilibrium dialysis are readily adaptable to high throughput but in the case of lipophilic drugs, being strongly bound to plasma proteins, their use is limited due to unspecific adsorption. Due to the fact that in recent years a trend to more lipophilic drugs is observed4, the need for new techniques that overcome these problems and that can be adapted to high throughput is increasing.
One technique that was especially designed for the determination of protein binding of lipophilic drugs is based on the distribution of drugs between plasma and erythrocytes or buffer and erythrocytes, respectively5. In the following, this technique is referred to as the partitioning method. Unfortunately, precision of the basic method is poor in the case of highly protein bound drugs (i.e., in the case of drugs which show high affinity to proteins).
A modification of the partitioning method is known to the person skilled in the art6, which overcomes that disadvantage by determining fu at several dilutions of plasma via linear regression. Another modification of the partition method circumvents the most critical step in the determination of fu via partitioning, the handling of the drug in protein free medium7.
It is known that Transil® is a widely used substance for the high throughput determination of membrane affinities in drug discovery9,10. A person skilled in the art will recognise that Transil® comprises solid silica particles that are coated with egg yolk phosphatidylcholine.
Recently a new approach for the determination of relative free fractions by equilibrium dialysis was reported using plasma of different species in each dialysis chamber16. However, validation of this approach is still outstanding. Own experiments using this method for drugs highly bound to plasma proteins (fu<0.5%) did not yield valid results (data not shown).
The closest prior art discloses a modification of the partitioning method that uses diluted plasma from various species but only erythrocytes from a single species8, thereby circumventing the necessity to isolate fresh erythrocytes for each individual test organism being under investigation in cross-species studies.
The new method is an advancement of the previously described erythrocytes partitioning technique8. The most time-consuming step in this method is the preparation of the erythrocytes: they are obtained by centrifugation of fresh, heparinised blood and have to be washed three times in isotonic phosphate buffer. Furthermore, the washing of the erythrocytes has to be done very carefully to avoid hemolysis. However, in some cases hemolysis can not be completely prevented and erroneous results due to binding of drugs to hemoglobin can not be excluded. All these difficulties are avoided using solid-supported lipid membranes. The material is commercially available and was especially developed to determine membrane affinities in HTS format (as an alternative to liposomes)9,10.